Key features and details
- Mouse monoclonal [DE2B4] to Amyloid Precursor Protein
- Suitable for: WB, ICC, IP, IHC-P
- Knockout validated
- Reacts with: Cow, Human, Pig, Monkey
- Isotype: IgG1
Product nameAnti-Amyloid Precursor Protein antibody [DE2B4]
See all Amyloid Precursor Protein primary antibodies
DescriptionMouse monoclonal [DE2B4] to Amyloid Precursor Protein
SpecificityReacts with beta-amyloid peptide and secreted amyloid precursor protein (alpha-secretase cleaved). This antibody has very weak recognition of full length APP and we do not recommend it for this purpose. It does however strongly recognize alpha-secretase cleaved soluble APP as well as b-amyloid peptide (40 and 42), in both cases native an denatured forms.
Tested applicationsSuitable for: WB, ICC, IP, IHC-Pmore details
Species reactivityReacts with: Cow, Human, Pig, Monkey
Does not react with: Mouse, Rat
Synthetic peptide corresponding to Human Amyloid Precursor Protein aa 672-689 conjugated to keyhole limpet haemocyanin. Corresponding to amino acids 1-16 of amyloid beta protein.
EpitopeAb12266 recognizes an epitope within amino acids 1-17 of the amyloid beta/A4 peptide region of APP. As such it recognises the C-terminus of alpha-secretase cleaved APP and amyloid beta/A4 peptide. It does not recognize the signal peptide of APP.
- WB: HAP1, HepG2, SH-SY5Y, HEK-293T and HeLa cell lysates.
General notesPlease note this antibody is very susceptible to freezing. Do NOT freeze.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C or -80°C. Avoid freeze / thaw cycle.
Storage bufferPreservative: 0.01% Sodium azide
Concentration information loading...
PurityTissue culture supernatant
KO cell lysates
Our Abpromise guarantee covers the use of ab12266 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/50 - 1/500.|
|ICC||Use at an assay dependent concentration. Use ice-cold methanol fixation for 10 minutes, followed by a brief wash with TBS. We have also used 4% paraformaldehyde for 20 minutes followed by permeabilization with either methanol of 0.5% Triton X-100 in TBS and this also seems to work well.|
|IP||Use at an assay dependent concentration.|
|IHC-P||Use at an assay dependent concentration. Retrieve antigens with 70% Formic acid for 10-30 minutes at room temperature before commencing with IHC staining protocol. Please note: it is important if ab12266 is being used to stain amyloid plaques in Alzheimer's disease brains that the sections are pre-treated with formic acid. This requirement applies to most, if not all amyloid antibodies and is not unique to this product.|
FunctionFunctions as a cell surface receptor and performs physiological functions on the surface of neurons relevant to neurite growth, neuronal adhesion and axonogenesis. Involved in cell mobility and transcription regulation through protein-protein interactions. Can promote transcription activation through binding to APBB1-KAT5 and inhibits Notch signaling through interaction with Numb. Couples to apoptosis-inducing pathways such as those mediated by G(O) and JIP. Inhibits G(o) alpha ATPase activity (By similarity). Acts as a kinesin I membrane receptor, mediating the axonal transport of beta-secretase and presenilin 1. Involved in copper homeostasis/oxidative stress through copper ion reduction. In vitro, copper-metallated APP induces neuronal death directly or is potentiated through Cu(2+)-mediated low-density lipoprotein oxidation. Can regulate neurite outgrowth through binding to components of the extracellular matrix such as heparin and collagen I and IV. The splice isoforms that contain the BPTI domain possess protease inhibitor activity. Induces a AGER-dependent pathway that involves activation of p38 MAPK, resulting in internalization of amyloid-beta peptide and leading to mitochondrial dysfunction in cultured cortical neurons. Provides Cu(2+) ions for GPC1 which are required for release of nitric oxide (NO) and subsequent degradation of the heparan sulfate chains on GPC1.
Beta-amyloid peptides are lipophilic metal chelators with metal-reducing activity. Bind transient metals such as copper, zinc and iron. In vitro, can reduce Cu(2+) and Fe(3+) to Cu(+) and Fe(2+), respectively. Beta-amyloid 42 is a more effective reductant than beta-amyloid 40. Beta-amyloid peptides bind to lipoproteins and apolipoproteins E and J in the CSF and to HDL particles in plasma, inhibiting metal-catalyzed oxidation of lipoproteins. Beta-APP42 may activate mononuclear phagocytes in the brain and elicit inflammatory responses. Promotes both tau aggregation and TPK II-mediated phosphorylation. Interaction with overexpressed HADH2 leads to oxidative stress and neurotoxicity. Also binds GPC1 in lipid rafts.
Appicans elicit adhesion of neural cells to the extracellular matrix and may regulate neurite outgrowth in the brain.
The gamma-CTF peptides as well as the caspase-cleaved peptides, including C31, are potent enhancers of neuronal apoptosis.
N-APP binds TNFRSF21 triggering caspase activation and degeneration of both neuronal cell bodies (via caspase-3) and axons (via caspase-6).
Tissue specificityExpressed in all fetal tissues examined with highest levels in brain, kidney, heart and spleen. Weak expression in liver. In adult brain, highest expression found in the frontal lobe of the cortex and in the anterior perisylvian cortex-opercular gyri. Moderate expression in the cerebellar cortex, the posterior perisylvian cortex-opercular gyri and the temporal associated cortex. Weak expression found in the striate, extra-striate and motor cortices. Expressed in cerebrospinal fluid, and plasma. Isoform APP695 is the predominant form in neuronal tissue, isoform APP751 and isoform APP770 are widely expressed in non-neuronal cells. Isoform APP751 is the most abundant form in T-lymphocytes. Appican is expressed in astrocytes.
Involvement in diseaseAlzheimer disease 1
Cerebral amyloid angiopathy, APP-related
Sequence similaritiesBelongs to the APP family.
Contains 1 BPTI/Kunitz inhibitor domain.
DomainThe basolateral sorting signal (BaSS) is required for sorting of membrane proteins to the basolateral surface of epithelial cells.
The NPXY sequence motif found in many tyrosine-phosphorylated proteins is required for the specific binding of the PID domain. However, additional amino acids either N- or C-terminal to the NPXY motif are often required for complete interaction. The PID domain-containing proteins which bind APP require the YENPTY motif for full interaction. These interactions are independent of phosphorylation on the terminal tyrosine residue. The NPXY site is also involved in clathrin-mediated endocytosis.
modificationsProteolytically processed under normal cellular conditions. Cleavage either by alpha-secretase, beta-secretase or theta-secretase leads to generation and extracellular release of soluble APP peptides, S-APP-alpha and S-APP-beta, and the retention of corresponding membrane-anchored C-terminal fragments, C80, C83 and C99. Subsequent processing of C80 and C83 by gamma-secretase yields P3 peptides. This is the major secretory pathway and is non-amyloidogenic. Alternatively, presenilin/nicastrin-mediated gamma-secretase processing of C99 releases the amyloid beta proteins, amyloid-beta 40 (Abeta40) and amyloid-beta 42 (Abeta42), major components of amyloid plaques, and the cytotoxic C-terminal fragments, gamma-CTF(50), gamma-CTF(57) and gamma-CTF(59). Many other minor beta-amyloid peptides, beta-amyloid 1-X peptides, are found in cerebral spinal fluid (CSF) including the beta-amyloid X-15 peptides, produced from the cleavage by alpha-secretase and all terminating at Gln-686.
Proteolytically cleaved by caspases during neuronal apoptosis. Cleavage at Asp-739 by either caspase-6, -8 or -9 results in the production of the neurotoxic C31 peptide and the increased production of beta-amyloid peptides.
N- and O-glycosylated. O-glycosylation on Ser and Thr residues with core 1 or possibly core 8 glycans. Partial tyrosine glycosylation (Tyr-681) is found on some minor, short beta-amyloid peptides (beta-amyloid 1-15, 1-16, 1-17, 1-18, 1-19 and 1-20) but not found on beta-amyloid 38, beta-amyloid 40 nor on beta-amyloid 42. Modification on a tyrosine is unusual and is more prevelant in AD patients. Glycans had Neu5AcHex(Neu5Ac)HexNAc-O-Tyr, Neu5AcNeu5AcHex(Neu5Ac)HexNAc-O-Tyr and O-AcNeu5AcNeu5AcHex(Neu5Ac)HexNAc-O-Tyr structures, where O-Ac is O-acetylation of Neu5Ac. Neu5AcNeu5Ac is most likely Neu5Ac 2,8Neu5Ac linked. O-glycosylations in the vicinity of the cleavage sites may influence the proteolytic processing. Appicans are L-APP isoforms with O-linked chondroitin sulfate.
Phosphorylation in the C-terminal on tyrosine, threonine and serine residues is neuron-specific. Phosphorylation can affect APP processing, neuronal differentiation and interaction with other proteins. Phosphorylated on Thr-743 in neuronal cells by Cdc5 kinase and Mapk10, in dividing cells by Cdc2 kinase in a cell-cycle dependent manner with maximal levels at the G2/M phase and, in vitro, by GSK-3-beta. The Thr-743 phosphorylated form causes a conformational change which reduces binding of Fe65 family members. Phosphorylation on Tyr-757 is required for SHC binding. Phosphorylated in the extracellular domain by casein kinases on both soluble and membrane-bound APP. This phosphorylation is inhibited by heparin.
Extracellular binding and reduction of copper, results in a corresponding oxidation of Cys-144 and Cys-158, and the formation of a disulfide bond. In vitro, the APP-Cu(+) complex in the presence of hydrogen peroxide results in an increased production of beta-amyloid-containing peptides.
Trophic-factor deprivation triggers the cleavage of surface APP by beta-secretase to release sAPP-beta which is further cleaved to release an N-terminal fragment of APP (N-APP).
Beta-amyloid peptides are degraded by IDE.
Cellular localizationMembrane. Membrane, clathrin-coated pit. Cell surface protein that rapidly becomes internalized via clathrin-coated pits. During maturation, the immature APP (N-glycosylated in the endoplasmic reticulum) moves to the Golgi complex where complete maturation occurs (O-glycosylated and sulfated). After alpha-secretase cleavage, soluble APP is released into the extracellular space and the C-terminal is internalized to endosomes and lysosomes. Some APP accumulates in secretory transport vesicles leaving the late Golgi compartment and returns to the cell surface. Gamma-CTF(59) peptide is located to both the cytoplasm and nuclei of neurons. It can be translocated to the nucleus through association with APBB1 (Fe65). Beta-APP42 associates with FRPL1 at the cell surface and the complex is then rapidly internalized. APP sorts to the basolateral surface in epithelial cells. During neuronal differentiation, the Thr-743 phosphorylated form is located mainly in growth cones, moderately in neurites and sparingly in the cell body. Casein kinase phosphorylation can occur either at the cell surface or within a post-Golgi compartment. Associates with GPC1 in perinuclear compartments. Colocalizes with SORL1 in a vesicular pattern in cytoplasm and perinuclear regions.
- Information by UniProt
- A4 amyloid protein antibody
- A4_HUMAN antibody
- AAA antibody
Lanes 1-4 : Anti-Amyloid Precursor Protein antibody [DE2B4] (ab12266) at 1/500 dilution
Lanes 5-8 : Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) at 1/20000 dilution
Lanes 1 & 5 : HeLa cell lysate
Lanes 2 & 6 : SH-SY5Y cell lysate
Lanes 3 & 7 : Wild-type HEK-293T cell lysate
Lanes 4 & 8 : APP knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Lanes 1 - 4: Green signal. Green - ab12266 observed at 110 kDa.
Lanes 5 - 8: Red signal. Red - loading control, ab181602 observed at 37 kDa.
ab12266 was shown to react with Amyloid Precursor Protein in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab255362 (knockout cell lysate ab263777) was used. Wild-type and Amyloid Precursor Protein knockout samples were subjected to SDS-PAGE. ab12266 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes : Anti-Amyloid Precursor Protein antibody [DE2B4] (ab12266) at 1/50 dilution
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Amyloid Precursor Protein knockout HAP1 whole cell lysate
Lane 3 : HepG2 whole cell lysate
Lane 4 : HeLa whole cell lysate
Lysates/proteins at 20 µg per lane.
Lanes 1 - 4: Merged signal (red and green). Green - ab12266 observed at 110 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab12266 was shown to specifically react with Amyloid Precursor Protein when Amyloid Precursor Protein knockout samples were used. Wild-type and Amyloid Precursor Protein knockout samples were subjected to SDS-PAGE. ab12266 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/50 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry of formaldehyde fixed brain tissue, after antigen-retrieval by formic acid treatment, for post-mortem diagnosis of Alzheimer s disease.
A section from the temporal lobe of a patient who died with Alzheimer’s disease was pre-treated with formic acid for 10 minutes prior to reaction with mouse monoclonal antibody DE4B2 (ab12266) that recognises an epitope near the N-terminus of
β-amyloid peptides. Bound antibody was detected by reaction with alkaline phosphatase-conjugated goat ant mouse IgG followed by BCIP/NBT substrate. Plaques in the neuropil have been circled in black. Amyloid deposits stained by DE2B4 (ab12266) can be seen around some blood vessels (*) but some are not stained (#)
IHC image of ab12266 staining in human Alzheimer brain (cerebral cortex) formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab12266, at a 1 in 40 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ab12266 has been referenced in 6 publications.
- Zhang W et al. Knockdown of BACE1-AS by siRNA improves memory and learning behaviors in Alzheimer's disease animal model. Exp Ther Med 16:2080-2086 (2018). PubMed: 30186443
- Kim J et al. HIV-1 Tat Interacts with and Regulates the Localization and Processing of Amyloid Precursor Protein. PLoS One 8:e77972 (2013). IP ; Human . PubMed: 24312169
- Fatemi SH et al. Impairment of fragile X mental retardation protein-metabotropic glutamate receptor 5 signaling and its downstream cognates ras-related C3 botulinum toxin substrate 1, amyloid beta A4 precursor protein, striatal-enriched protein tyrosine phosphatase, and homer 1, in autism: a postmortem study in cerebellar vermis and superior frontal cortex. Mol Autism 4:21 (2013). WB ; Human . PubMed: 23803181
- Cagnin M et al. Dopamine induces apoptosis in APPswe-expressing Neuro2A cells following Pepstatin-sensitive proteolysis of APP in acid compartments. Brain Res 1471:102-17 (2012). WB . PubMed: 22771396
- Pavlov PF et al. Mitochondrial ?-secretase participates in the metabolism of mitochondria-associated amyloid precursor protein. FASEB J 25:78-88 (2011). PubMed: 20833873
- Chiarugi A & Moskowitz MA Poly(ADP-ribose) polymerase-1 activity promotes NF-kappaB-driven transcription and microglial activation: implication for neurodegenerative disorders. J Neurochem 85:306-17 (2003). PubMed: 12675907